Method for increasing the concentration of ascorbic acid in brain tissues of a subject

ABSTRACT

This invention provides a method for increasing the concentration of ascorbic acid in the cells of a subject which comprises administering to the subject an amount of dehydroascorbic acid effective to increase the concentration of ascorbic acid in the subject&#39;s cells. This invention further provides a method for increasing the antioxidant potential of the cells of a subject which comprises administering to the subject an amount of dehydroascorbic acid effective to increase the antioxidant potential of the subject&#39;s cells. This invention also provides a method for increasing the concentration of ascorbic acid in brain tissue of a subject which comprises administering to the subject an amount of dehydroascorbic acid effective to increase the concentration of ascorbic acid in the subject&#39;s brain tissue. This invention also provides a method for increasing the antioxidant potential of brain tissue of a subject which comprises administering to the subject an amount of dehydroascorbic acid effective to increase the antioxidant potential of the subject&#39;s brain tissue.

[0001] This application is a continuation-in-part of U.S. Ser. No. NotYet Known, filed Nov. 19, 1999, which is a continuation of PCTInternational Application No. PCT/US98/10608, filed May 21, 1998,claiming priority of of U.S. Provisional Application Nos. 60/067,185,filed Dec. 1, 1997 and 60/047,271, filed May 21, 1997, the contents ofwhich are hereby incorporated by reference into this application.

[0002] This invention was made with support under Grant Nos. RO1 CA30388and RO1 HL42107 from the National Institutes of Health. Accordingly, theUnited States Government has certain rights in the invention.

[0003] Throughout this application, various references are referred toby arabic numerals within parentheses. Disclosures of these publicationsin their entireties are hereby incorporated by reference into thisapplication to more fully describe the state of the art to which thisinvention pertains. Full bibliographic citation for these references maybe found at the end the specification, immediately preceding the claims.

BACKGROUND OF THE INVENTION

[0004] Numerous connections have been made between the generation andpresence of oxidative free radicals in brain tissue and neurologicaldisorders. For example, 1) Jenner (26) links oxidative stress toParkinson's, Alzheimer's and Huntington's diseases. 2) Recent clinicalstudies have demonstrated that alpha-tocopherol (vitamin E) andselegiline (deprenyl), pharmacologic agents that have antioxidantactivity, can slow the progression of moderately severe Alzheimer'sdisease (27). 3) Antioxidants such as vitamin C and vitamin E may havean important role in the treatment of diseases whose pathogenesisinvolves free radical formation and impaired antioxidant defenses in theaging population. Oxidative damage has been hypothesized as central tothe neurodegenerative processes such as Alzheimer's disease (28).According to the free radical hypothesis, Alzheimer's disease is anacceleration of the normal aging process in affected brain regions whichbecome progressively more damaged by free radicals generated frommetabolism. In Alzheimer's disease, the cerebral cortex seems to haveincreased antioxidant requirements, increased sensitivity to freeradicals, and levels of the free radical defense enzymes, such assuperoxide dismutase, that are reduced by 25-35% in the frontal cortexand hippocampus. The loss of hippocampal cholinergic neurons is a keyfeature of Alzheimer's disease and these neurons seem particularlyvulnerable to the deleterious effects of free radicals on the muscariniccholinergic receptor (29). 4) Antioxidants have been tested as drugs forParkinson's disease (30), and it was found that selegiline, which mayact as an antioxidant since it inhibits oxidative deamination, delaysthe onset of the disability (31). 5) Peyser et al. concluded thatantioxidant therapy may slow the rate of motor decline early in thecourse of Huntington's disease (35). 6) According to Challem (32) freeradicals and oxidative stress may be factors involved with thepathogenesis of Mad Cow disease. 7) The oxidative modification oflow-density lipoprotein (LDL), termed lipid peroxidation has been shownto be an initiating event in atherosclerosis. Probucol, an antioxidant,is effective in reducing the rate of restenosis after balloon coronaryangioplasty (36). Oxidized LDL has several detrimental effects on cellsincluding brain cells such as cytotoxicity and vascular dysfunction.

[0005] Therefore, increasing the concentration of free-radicalscavengers or antioxidants in brain tissue may provide therapeuticbenefits to subjects suffering from neurodegenerative diseases. Sano etal. conclude (27) that the use of the antioxidants, selegiline orvitamin E may delay clinically important functional deterioration inpatients with Alzheimer's disease. Their results are particularlysignificant because vitamin E does not cross the blood-brain barrier inlarge amounts, and still it has a measurable effect.

[0006] The enhancement of the antioxidant potential is useful intreating of many diseases. For example, the increase of antioxidantpotential achieved by this invention will be able to treat stroke andneurovascular diseases. It is known that ischemic stroke is the mostcommon neurologic disorder causing death or disability among adults.Strokes of all types rank third as a cause of death, surpassed only byheart disease and cancer. Ischemic stroke events account forapproximately 85% of all strokes. Because no medical or surgicaltreatment has yet been established as reversing the effects of acuteischemic stroke, early identification and treatment of persons at thetime they present with stroke is compelling, if such a treatment isefficacious. Currently, there are no approved treatments for stroke. Thedamage from stroke is caused by occlusion of a vessel, therebyrestricting the delivery of oxygen in the blood to an area of the brain.Much of the damage is caused by damage from oxygen free radicals in thearea served by the occluded vessel after reperfusion of the affectedarea (37). Thus, increasing the antioxidant potential of the brain mayhave beneficial effects on stroke and other neurovascular diseases.

[0007] Therefore, increasing vitamin C concentrations in the brain byproviding dehydroascorbic acid to the subject could enhance antioxidantpotential in the central nervous system and may be therapeutic in strokeand neurovascular diseases as described.

[0008] Researchers have proposed that atherosclerosis, and its deadlyeffects of heart attack and stroke, develops in relationship tooxidation of low-density lipoproteins (LDL) carrying cholesterol in theblood. The theory states that free radicals generated by the body's ownimmune cells oxidize LDL which is taken up by cells of the vascularintima initiating the atherosclerosis lesion. Ultraviolet and gammaradiation, cigarette smoke and other environmental pollutants, alsocause oxidative damage to cells and vital compounds. The damage leads tothe development of several chronic diseases including cancer andcoronary heart disease (CHD). It was further proposed that antioxidantssuch as vitamin E and C and the carotenoids could prevent damage and theensuing diseases. Many epidemiologic and animal studies have offeredevidence to support the theory (33, 34). Recent studies demonstratedthat the antioxidant proburol is effective in reducing the rate ofrestenosis after balloon coronary angioplasty (36).

[0009] Evidence suggests that the neuropathology of Huntington'sdisease, a neuropsychiatric disorder, results from excessive activationof glutamate-gated ion channels, which kills neurons by oxidativestress. It was reported that antioxidant therapy may slow the rate ofmotor decline early in the course of Huntington's disease (35). VitaminC enters cells, in vi tro, through the facilitative glucose transporterGLUT1 in the form of dehydroascorbic acid and is retainedintracellularly as ascorbic acid (1). In order to test the hypothesisthat GLUT1 transport of dehydroascorbic acid is a primary physiologicalmechanism for tissue acquisition of vitamin C, we investigated thetransport of vitamin C across the blood-brain barrier (BBB) in rodents.GLUT1 is expressed at the BBB on endothelial cells and is responsiblefor glucose entry into the brain. Ascorbic acid, the predominant form ofvitamin C in blood, was incapable of crossing the BBB whiledehydroascorbic acid readily entered the brain and was retained in theform of ascorbic acid. The transport of dehydroascorbic acid into thebrain was competitively inhibited by D-glucose, but not by L-glucose.These findings define the transport of dehydroascorbic acid by GLUT1 asthe mechanism by which the brain acquires vitamin C, and point to theoxidation of vitamin C as the important regulatory step in theaccumulation of the vitamin by the brain.

[0010] Dehydroascorbic acid, the oxidized form of vitamin C, waspreviously found to be transported through the facilitative glucosetransporters. Expression of GLUT1, GLUT2, and GLUT4 in Xenopus oocytesconferred the ability to take up dehydroascorbic acid which was retainedintracellularly after it was reduced to ascorbic acid (1). It was alsoestablished that facilitative glucose transporters are involved in thetransport and accumulation of vitamin C by normal human neutrophils andthe myeloid leukemia cell line, HL60 (1-3). In these cellsdehydroascorbic acid is transported across the cell membrane andaccumulated in the reduced form, ascorbic acid, which is nottransportable through the bidirectional glucose transporter (1-3).Ascorbic acid may be transported through a Na⁺-ascorbate co-transporterthat is reported to be present in small intestine, kidney andadrenomedullary chromaffin cells (4). The co-transporter has not beenmolecularly characterized and no Na⁺-dependent ascorbic acid uptake inwhite blood cells has been found (2,3).

[0011] GLUT1 is expressed on endothelial cells at the BBB and isresponsible for glucose transport into the brain (5,6). In the 1880's,Ehrlich found that intravenously injected aniline dyes colored all ofthe organs of experimental rabbits except the brain and the spinal cord(7,8). This observation led to the eventual discovery that the BBB iscomprised of a wall of capillaries forming an endothelial barrierbetween the blood and the brain, functioning primarily to regulate thetransport of nutrients and waste products (9,10). Several nutrienttransporters have been identified at the BBB including GLUT1, amonocarboxylic acid transporter, neutral amino acid transporter, aminetransporter, basic amino acid transporter, nucleoside transporter, andpurine base transporter (11). Here it is shown in rodents that vitamin Ccrosses the BBB through GLUT1 only in the oxidized form, dehydroascorbicacid, and is retained in the brain in the reduced form, ascorbic acid.

[0012] The present invention allows for the controlled introduction ofthe antioxidant vitamin C into brain tissue, which should serve as animportant therapeutic method to treat and prevent various disordersassociated with free radicals and oxidative damage.

SUMMARY OF THE INVENTION

[0013] This invention provides a method for increasing the concentrationof ascorbic acid in a cell which comprises contacting the cell with anamount of dehydroascorbic acid effective to increase the concentrationof ascorbic acid in the cell. This invention also provides a method forincreasing the antioxidant potential in a cell which comprisescontacting the cell with an amount of dehydroascorbic acid effective toincrease the antioxidant potential in the cell.

[0014] In one embodiment, the cell is a brain cell. In one embodiment,the cell is present in a tissue. In one embodiment, the tissue is abrain tissue. This invention provides the above method wherein the cellis present in a subject and the contacting is effected by administeringthe dehydroascorbic acid to the subject.

[0015] This invention also provides a method for increasing theconcentration of ascorbic acid in the cells of a subject which comprisesadministering to the subject an amount of dehydroascorbic acid effectiveto increase the concentration of ascorbic acid in the subject's cells.This invention further provides a method for increasing the antioxidantpotential of the cells of a subject which comprises administering to thesubject an amount of dehydroascorbic acid effective to increase theantioxidant potential of the subject's cells.

[0016] This invention provides a method for increasing the concentrationof ascorbic acid in brain tissue of a subject which comprisesadministering to the subject an amount of dehydroascorbic acid effectiveto increase the concentration of ascorbic acid in the subject's braintissue.

[0017] This invention also provides a method for increasing theantioxidant potential of brain tissue of a subject which comprisesadministering to the subject an amount of dehydroascorbic acid effectiveto increase the antioxidant potential of the subject's brain tissue.This invention provides a method for treating or preventing dementia ina subject comprising administering to the subject an amount ofdehydroascorbic acid effective to increase the concentration of ascorbicacid in the subject's brain tissue so as to thereby treat or preventdementia in the subject.

[0018] This invention provides a method for treating or preventingdementia in a subject comprising administering to the subject an amountof dehydroascorbic acid effective to increase the antioxidant potentialof the subject's brain tissue so as to thereby treat or prevent dementiain the subject.

[0019] This invention also provides a method for treating or preventingdiseases involving free radicals in a subject comprising administeringto the subject an amount of dehydroascorbic acid effective to increasethe concentration of ascorbic acid in the subject's cells so as tothereby treat or prevent diseases involving free radicals in thesubject.

[0020] This invention also provides a method for treating or preventingdiseases involving free radicals in a subject comprising administeringto the subject an amount of dehydroascorbic acid effective to increasethe antioxidant potential of cells so as to thereby treat or preventdiseases involving free radicals in the subject.

[0021] This invention also provides a method of prophylaxis for thesediseases. The diseases include but are not limited to cancer, acardiovascular disease and cataracts.

[0022] This invention also provides a method for slowing the agingprocess of a subject comprising administering to the subject an amountof dehydroascorbic acid effective to increase the antioxidant potentialof cells so as to thereby slow the aging process in the subject.

[0023] This invention also provides a method for treating a subjectinfected with human immunodeficiency virus comprising administering tothe subject an amount of dehydroascorbic acid effective to treat thesubject infected with human immunodeficiency virus.

[0024] This invention provides a method for treating or preventing aneurodegenerative disease in a subject comprising administering to thesubject an amount of dehydroascorbic acid effective to increase theconcentration of ascorbic acid in the subject's brain tissue so as tothereby treat or prevent a neurodegenerative disease in the subject.

[0025] This invention also provides a method for treating or preventinga neurodegenerative disease in a subject comprising administering to thesubject an amount of dehydroascorbic acid effective to increase theantioxidant potential of the subject's brain tissues so as to therebytreat or prevent a neurodegenerative disease in the subject.

[0026] This invention provides a method for treating or preventingstroke or neurovascular disease or other diseases which can be caused bylipid peroxidation in a subject comprising administering to the subjectan amount of dehydroascorbic acid effective to increase theconcentration of ascorbic acid in the subject's brain tissue so as tothereby treat or prevent stroke or neurovascular disease or otherdiseases which can be caused by lipid peroxidation in the subject.

[0027] This invention also provides a method for treating or preventingstroke or neurovascular disease or other diseases which can be caused bylipid peroxidation in a subject comprising administering to the subjectan amount of dehydroascorbic acid effective to increase the antioxidantpotential of the brain tissues so as to thereby treat or prevent strokeor neurovascular disease or other diseases which can be caused by lipidperoxidation in the subject.

[0028] Moreover, this invention provide a method for treating orpreventing central nervous system manifestations of genetic diseases.

[0029] This invention provides a method for preventing or treatingbehavioral disorders in a subject comprising administering to thesubject an amount of dehydroascorbic acid effective to increase theconcentration of ascorbic acid in the subject's brain tissue so as tothereby prevent or treat behavioral disorders in the subject.

[0030] This invention also provides a method for preventing or treatingbehavioral disorders in a subject comprising administering to thesubject an amount of dehydroascorbic acid effective to increase theantioxidant potential of the subject's brain tissue so as to therebyprevent or treat behavioral disorders in the subject.

[0031] This invention provides the above methods which further compriseadministering to the subject a therapeutically effective amount of asecond agent.

BRIEF DESCRIPTION OF THE FIGURES

[0032]FIG. 1 Dehydroascorbic acid is transported across the BBB andaccumulates in the brain as ascorbic acid. (A) Balb/c mice (age 6-8weeks) and (B) Fischer F344 rats (70-80 gram body weight) were injectedinto the tail vein with 5 μCi(mouse) or 10 μCi(rat) ¹⁴C-ascorbic acid(L-[1-¹⁴C]-ascorbic acid, specific activity, 6.6 mCi/mmol, Dupont NEN),¹⁴C-dehydroascorbic acid or ³H-sucrose ([fructose-1-³H]-sucrose,specific activity 20.0 Ci/mmol, Dupont NEN). Each group consists of 12animals and the values are expressed as mean±SEM. (C)HPLC analysis ofthe methanol soluble fraction of the brain and (H) serum of a mouseinjected with 20 μCi ¹⁴C-dehydroascorbic acid and sacrificed at 5 min(injected material, hashed line). (C) Accumulation of vitamin C in thebrain is in the form of ascorbic acid (˜90 &; retention time≈11.80 min,solid line). (H) Radioactivity present in serum is in the form ofascorbic acid (>98%; retention time≈11.80 min, solid line). (D) Theinitial kinetics and (E) 2 hr kinetics of accumulation of radioactivityin the brain of mice injected intravenously with ¹⁴C-ascorbic acid (),¹⁴C-dehydroascorbic acid (▪) or ³H-sucrose (◯). (F) The initial kineticsand (G) 2 hr kinetics of radioactivity in the serum of mice injectedintravenously with ¹⁴C-ascorbic acid (), ¹⁴C-dehydroascorbic acid (▪)or ³H-sucrose (◯). Each data set in (D) through (G) represents 4mice±SEM.

[0033]FIG. 2 Specificity of the transport of dehydroascorbic acidthrough GLUT1 at the Balb/c mouse BBB. (A) ¹⁴C-Dehydroascorbic acid (▪)entered the brain and its accumulation was blocked by increasing amountsof D-deoxyglucose which is transported through GLUT1. Transport of³H-leucine (◯) or ¹⁴C-ascorbic acid () across the BBB was not affectedby D-deoxyglucose. (B) L-glucose, which is not transported throughGLUT1, had no effect on the transport of ¹⁴C-dehydroascorbic acid.Transport of ³H-leucine (◯) or ¹⁴C-ascorbic acid () across the BBB wasnot affected by L-glucose. All experiments were carried out over a30-second time course. Each data set included 4 mice and the data wereexpressed as mean±SEM. A mouse has a baseline serum glucoseconcentration of approximately 12 mM, which calculates to 2.67 mgglucose in the entire mouse based on the average plasma volume of themouse. The amount of exogenous glucose administered in this experimentwas based on this number and subsequent multiples to a maximum tolerablelevel.

[0034]FIG. 3 Brain digital autoradiography of rat with ¹⁴C-labeledascorbic acid, dehydroascorbic acid, D-deoxyglucose and sucrose. (A)Digital autoradiography was performed on a Fisher F344 rat (8 wks ofage) 3 min after intravenous injection with 40 μCi of¹⁴C-dehydroascorbic acid, (B) 40 μCi ¹⁴C-ascorbic acid and (C)40 μCi¹⁴C-sucrose ([glucose-¹⁴C(U)]-sucrose, specific activity, 310 mCi/mmol,Dupont NEN). The area of the brain is denoted with an * in the figure.The photo-stimulated luminescence (PSL)/mm² ratio of brain/backgroundcounts for the dehydroascorbic acid-injected rat was 8.6±0.3 (mean of 3sections±SEM). The PSL/mm² ratio in the ascorbic acid-injected rat was1.5±0.1 and 1.4±0.1 in the sucrose-injected rat.

[0035]FIG. 7 Tracer studies revealed accumulation of nearly 4% of theDHA (expressed as percent of injected dose (TD) per gram of braintissue) compared to only trace levels of ascorbic acid and sucrose inischemic animals.

[0036]FIG. 8

[0037] A Both high (250 mg/kg) and low dose (40 mg/kg) DHA pre-treatmentof animals undergoing reperfused stroke, demonstrated a dose-dependentimprovement in post-ischemic cerebral perfusion compared to both sucroseand ascorbic acid treated animals (19±3% vehicle, 13±1% AA, 28±2%low-DHA, 40±4% high-DHA; p<0.05 for either DHA vs. either control).

[0038] B DHA conferred dose-dependent cerebroprotection as evidenced byboth decreased cerebral infarct volumes (54±6% vehicle, 58±4% AA, 22±4%low-DHA, 12±4% high-DHA; p<0.05 for either DHA vs. either control)

[0039] C Reductions in neurological deficit scores (4.0±0.2 vehicle,2.7±0.7 AA, 1.6±0.2 low-DHA, 1.9±0.2 high-DHA; p<0.05 for either DHA vs.vehicle).

[0040] D Ascorbic acid treatment was not associated with significantimprovements in either infarct size or neurological function, nor didascorbic acid treatment reduce overall mortality as was the case withDHA.

DETAILED DESCRIPTION OF THE INVENTION

[0041] This invention provides a method for increasing the concentrationof ascorbic acid in a cell which comprises contacting the cell with anamount of dehydroascorbic acid effective to increase the concentrationof ascorbic acid in the cell. This invention also provides a method forincreasing the antioxidant potential in a cell which comprisescontacting the cell with an amount of dehydroascorbic acid effective toincrease the antioxidant potential in the cell.

[0042] In one embodiment, the cell is a brain cell. In one embodiment,the cell is present in a tissue. In one embodiment, the tissue is abrain tissue. In one embodiment, the cell is present in a subject andthe contacting is effected by administering the dehydroascorbic acid tothe subject. In one embodiment, the subject is a human.

[0043] This invention also provides a method for increasing theconcentration of ascorbic acid in the cells of a subject which comprisesadministering to the subject an amount of dehydroascorbic acid effectiveto increase the concentration of ascorbic acid in the subject's cells.This invention further provides a method for increasing the antioxidantpotential of the cells of a subject which comprises administering to thesubject an amount of dehydroascorbic acid effective to increase theantioxidant potential of the subject's cells.

[0044] This invention provides a method for increasing the concentrationof ascorbic acid in brain tissue of a subject which comprisesadministering to the subject an amount of dehydroascorbic acid effectiveto increase the concentration of ascorbic acid in the subject's braintissue.

[0045] This invention also provides a method for increasing theantioxidant potential of brain tissue of a subject which comprisesadministering to the subject an amount of dehydroascorbic acid effectiveto increase the antioxidant potential of the subject's brain tissue.

[0046] There are several ways that dehydroascorbic acid can enter a cellof a tissue, one of which is through a facilitative glucose transporter.

[0047] The cells of the subject invention include but are not limited tobrain cells, neuronal cells, endothelial cells, glial cells, microglialcells, smooth muscle cells, somatic cells, bone marrow cells, livercells, intestinal cells, germ cells, myocytes, mononuclear phagocytes,tumor cells, and stem cells. The cell may also be another kind of cellsnot explicitly listed herein. In the preferred embodiment, the cells arebrain cells.

[0048] The subject may be a mammal or non-mammal. The subject may be ahuman, a primate, an equine, an opine, an avian, a bovine, a porcine, acanine, a feline, a murine, a mouse, a rat, or a cow. The subject mayalso be another kind of subject not explicitly listed here. In anotherembodiment, the subject is a vertebrate. In a preferred embodiment, themammal is a human being.

[0049] In one embodiment of the subject invention, the subject isafflicted with a neurodegenerative disease. Such neurodegenerativediseases include but are not limited to Alzheimer's Disease, Parkinson'sDisease or other forms of presenile dementia.

[0050] In one embodiment of the subject invention, the subject isafflicted with neurovascular disease. The neurovascular disease of thepresent invention includes but is not limited to stroke.

[0051] The subject may carry genetic diseases with central nervoussystem manifestations. In an embodiment, the genetic disease is theHuntington's disease.

[0052] In one embodiment of the present invention, the subject may beafflicted with a disease which involves the oxidative modification oflow-density lipoprotein peroxidase. These diseases include but are notlimited to stroke, atherosclerosis and neurodegenerative disorders.

[0053] In another embodiment, the human subject is afflicted with abehavioral disorder. Such behavioral disorders include but are notlimited to dysthymia, involution depression, aggressiveness viadominance, hyperactivity, deprivation syndrome, separation anxiety,intermittent anxiety, instrumental sociopathy, stereotypies, phobia or asocialization disorder. In a further embodiment, the subject isafflicted with schizophrenia.

[0054] This invention provides a method for treating or preventingdementia in a subject comprising administering to the subject an amountof dehydroascorbic acid effective to increase the concentration ofascorbic acid in the subject's brain tissue so as to thereby treat orprevent dementia in the subject.

[0055] This invention provides a method for treating or preventingdementia in a subject comprising administering to the subject an amountof dehydroascorbic acid effective to increase the antioxidant potentialof the subject's brain tissue so as to thereby treat or prevent dementiain the subject.

[0056] This invention also provides a method for treating or preventingdiseases involving free radicals in a subject comprising administeringto the subject an amount of dehydroascorbic acid effective to increasethe concentration of ascorbic acid in the subject's cells so as tothereby treat or prevent diseases involving free radicals in thesubject.

[0057] This invention also provides a method for treating or preventingdiseases involving free radicals in a subject comprising administeringto the subject an amount of dehydroascorbic acid effective to increasethe antioxidant potential of cells so as to thereby treat or preventdiseases involving free radicals in the subject.

[0058] This invention also provides a method of prophylaxis for thesediseases. The diseases include but are not limited to cancer, acardiovascular disease and cataracts.

[0059] These cancers include but are not limited to the following:prostate cancer; biliary tract cancer; brain cancer, includingglioblastomas and medelloblastomes; breast cancer; cervical cancer;choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer;gastric cancer; hematological neoplasms, including acute lymphocytic andmyelogenous leukemia, multiple myeloma, AIDS associated leukemias andadult T-cell leukemia lymphoma; intraepithelial neoplasms, includingBowens' disease and Paget's disease; liver cancer; lung cancer;lymphomas, including Hodgkin's disease and lymphozytic lymphomas;neuroblastomas; oral cancer, including squamous cell carcinoma; ovariancancer, including those arising from epithelial cells, stromal cells,germ cells and mesenchymal cells; pancreas cancer; rectal cancer;sarcomas, including leiomyosarcoma, rhabdomyosarcoma, liposarcoma,fibrosarcoma and osteosarcoma; skin cancer, including melanoma, Kaposi'ssarcoma, basocellular cancer and squamous cell cancer; testicularcancer, including terminal tumors (seminoma, non-seminoma (teratomas,choriocarcinomas)), stromal tumors and germ cell tumors; thyroid cancer,including thyroid adenocarcinoma and medullar carcinoma; and renalcancer including adenocarcinoma and Wilms tumor.

[0060] The cardiovascular conditions include but are not limited toartheresclerosis, post-myocardial infarction, stroke, post-angioplastyand an association with thrombolytic reperfusion.

[0061] The cataracts conditions include but are not limited to cornealopacification.

[0062] This invention also provides a method for slowing the agingprocess of a subject comprising administering to the subject an amountof dehydroascorbic acid effective to increase the antioxidant potentialof cells so as to thereby slow the aging process in the subject.

[0063] As used herein, “aging” means accumulation of oxidative damageover time.

[0064] This invention also provides a method for treating a subjectinfected with human immunodeficiency virus comprising administering tothe subject an amount of dehydroascorbic acid effective to treat thesubject infected with human immunodeficiency virus.

[0065] As used herein, human immunodeficiency virus can be abbreviatedas “HIV” and includes but is not limited to HIV-1. HIV includes but isnot limited to extracellular virus particles and the forms of HIV foundin HIV-1 infected cells. The modes of treatment include but are notlimited to inhibiting the growth of the virus, decreasing the ability ofthe virus to enter cells, and also decreasing T-cell deficiency.

[0066] As it will be easily appreciated by persons of skill in the art,this invention is applicable to both human and animal diseases whichcould be treated by antioxidants. This invention is intended to be usedin husbandry and veterinary medicine.

[0067] In this invention, the dehydroascorbic acid may be administeredorally, intravenously, subcutaneously, intramuscularly, topically, or byother routes or circumstances of administration by which thedehydroascorbic acid will not be hydrolyzed. Dehydroascorbic acidhydrolyses easily in aqueous solution. It is the intention of thisinvention to administer the dehydroascorbic acid in a stabilized form.It is known that dehydroascorbic acid is stable under low pH conditions.Accordingly, dehydroascorbic acid may be stored in low pH and thenadministered directly to a large vein of a subject. Alternatively,dehydroascorbic acid may be stored in powdered form and hydrated beforeadministering to a subject.

[0068] Moreover, dehydroascorbic acid may be encapsulated in liposomesat low pH. The encapsulated dehydroascorbic acid will then beadministered to a subject. In a preferred embodiment, the encapsulateddehydroascorbic acid is administered orally.

[0069] U.S. Pat. No. 4,822,816 describes uses of aldono-lactones andsalts of L-threonic, L-xylonic and L-lyxonic to stabilize thedehydroascorbic acid. The content of U.S. Pat. No. 4,822,816 is herebyincorporated into this application by reference. Accordingly, thismethod provides another means for stabilization of the dehydroascorbicacid.

[0070] Finally, appropriate amounts of ascorbic acid and ascorbateoxidase may be administered together to a subject to produce an amountof dehydroascorbic acid effective to increase the concentration ofascorbic acid in the brain tissues of the subject. Ascorbate oxidasecatalyzes oxidation of L-ascorbic acid, and it is commerciallyavailable. U.S Pat. No. 5,612,208 describes a new ascorbate oxidase andits gene, the content of which is hereby incorporated into thisapplication by reference. Accordingly, ascorbate oxidase may be producedby recombinant DNA technology.

[0071] Using this invention, the brain tissues of a subject may beloaded with the maximum amount of ascorbic acid.

[0072] Dehydroascorbic acid may exist in various salt forms. It is theintention of this invention to encompass these forms. The salts uponhydration will generate dehydroascorbic acid.

[0073] This invention provides a method for treating or preventing aneurodegenerative disease in a subject comprising administering to thesubject an amount of dehydroascorbic acid effective to increase theconcentration of ascorbic acid in the subject's brain tissue so as tothereby treat or prevent a neurodegenerative disease in the subject.

[0074] This invention also provides a method for treating or preventinga neurodegenerative disease in a subject comprising administering to thesubject an amount of dehydroascorbic acid effective to increase theantioxidant potential of the subject's brain tissues so as to therebytreat or prevent a neurodegenerative disease in the subject. Theneurodegenerative diseases include but are not limited to Alzheimer'sDisease and Parkinson's Disease.

[0075] This invention provides a method for treating or preventingstroke or neurovascular disease or other diseases which can be caused bylipid peroxidation in a subject comprising administering to the subjectan amount of dehydroascorbic acid effective to increase theconcentration of ascorbic acid in the subject's brain tissue so as tothereby treat or prevent stroke or neurovascular disease or otherdiseases which can be caused by lipid peroxidation in the subject.

[0076] This invention also provides a method for treating or preventingstroke or neurovascular disease or other diseases which can be caused bylipid peroxidation in a subject comprising administering to the subjectan amount of dehydroascorbic acid effective to increase the antioxidantpotential of the brain tissues so as to thereby treat or prevent strokeor neurovascular disease or other diseases which can be caused by lipidperoxidation in the subject.

[0077] These diseases include but are not limited to stroke,atherosclerosis and neurodegenerative disorders.

[0078] Moreover, this invention provides a method for treating orpreventing central nervous system manifestations of genetic diseases.The conditions of the disease will be improved by increasing theantioxidant potential of the brain. Prevention of such central nervoussystem manifestations of genetic disease may even be effected if theantioxidant potential of the brain is maintained at a high level. Thesegenetic diseases include but are not limited to Huntington's disease.

[0079] This invention provides a method for preventing or treatingbehavioral disorders in a subject comprising administering to thesubject an amount of dehydroascorbic acid effective to increase theconcentration of ascorbic acid in the subject's brain tissue so as tothereby prevent or treat behavioral disorders in the subject.

[0080] This invention also provides a method for preventing or treatingbehavioral disorders in a subject comprising administering to thesubject an amount of dehydroascorbic acid effective to increase theantioxidant potential of the subject's brain tissue so as to therebyprevent or treat behavioral disorders in the subject. Such behavioraldisorders include but are not limited to dysthymia, involutiondepression, aggressiveness via dominance, hyperactivity, deprivationsyndrome, separation anxiety, intermittent anxiety, instrumentalsociopathy, stereotypies, phobia or a socialization disorder. In anotherembodiment, the behavioral disorder is schizophrenia.

[0081] This invention provides the above methods which further compriseadministering to the subject a therapeutically effective amount of asecond agent.

[0082] When treating or preventing the behavioral disorders,dehydroascorbic acid may be used in combination with other drugs. Theymay be administered concomitantly or at different time points. Thisinvention also provides the above methods further comprisingadministering to the subject a therapeutically effective amount of asecond agent.

[0083] This invention also provides a combination therapy wherein aneffective amount of dehydroascorbic acid is administered withtherapeutic agents for the neurodegenerative disease. The administrationmay be performed concomitantly or at different time points. Whentreating the Alzheimer's disease, the therapeutic agents include, butare not limited to, Estrogen, Vitamin E (alpha-tocopherol), Tacrine(Tetrahydroacridinamine), Selegiline (Deprenyl), and Aracept(Donepezil). With respect to the Parkinson's disease, the therapeuticagents include, but are not limited to, the anticholinergic class ofdrugs, clozapine, levodopa with carbidopa or benserazide, Selegiline(Deprenyl), and dopamine agonist class of drugs.

[0084] This invention will be better understood from the ExperimentalDetails which follow. However, one skilled in the art will readilyappreciate that the specific methods and results discussed are merelyillustrative of the invention as described more fully in the claimswhich follow thereafter.

Experimental Details

[0085] Experimental Methods

[0086] Blood-brain barrier transport studies. ¹⁴C-dehydroascorbic acidwas generated in all experiments by incubating the ¹⁴C-ascorbic acidwith ascorbate oxidase, 1 unit/1.0 mmol L-ascorbate (derived fromCucurbita species, Sigma). Dithiothreitol (0.1 mmol/liter) was added tothe vitamin C preparations as a reducing agent. Animals were sacrificedat various time points after injection by cervical dislocation of CO₂inhalation. The brain was then dissected out and homogenized in 70%methanol. Samples were processed for scintillation spectrometry or HPLCas described (2,3). HPLC was performed on the methanol fraction with 1mmol/L EDTA added (2,3). Samples were stored at −70° C. until analysis.HPLC samples were separated on a Whatman strong anion exchange Partisil10 SAX (4.6×25-cm) column (Whatman, Hillsboro, Oreg.). A Whatman-typeWCS solvent-conditioning column was used and the eluates monitored witha Beckman System Gold liquid chromatograph (Beckman Instruments, Irvine,Calif.) with a diode array detector and radioisotope detector arrangedin series. Ascorbic acid was monitored by absorbance at 265 nm and byradioactivity. Dehydroascorbic acid shows no absorbance at 265 nm andwas monitored by radioactivity.

[0087] Digital autoradiography. Animals were sacrificed, frozen in a dryice/hexane mixture and then embedded in ˜5% carboxymethylcellulose(Sigma Aldrich). The animal blocks were allowed to equilibrate for —12hours at −20° C. and the animals were sectioned in coronal cuts with aslice thickness of ˜40-45 μm in a cryo-microtome (PMV), and tape liftedfor direct exposure onto digital plates (23). The exposure time wasapproximately 72 hours. All digital plates were scanned on a Fuji Bas5000 digital autoradiographic system (Fuji, Inc.) At 25 μm resolution.

[0088] Calculation of the BBB permeability-surface area product. Theamount of compound which crosses the BBB is dependent on two parametersdefined by the following equation: ${PS} = \frac{V_{D} - V_{0}}{t}$

[0089] where PS is the BBB permeability-surface area product and AUC isthe plasma area under the concentration time-activity curve at a giventime (t) after injection. A variant of the single intravenous injectiontechnique termed the external organ technique was used to quantify theBBB PS product in anesthetized animals. The plasma and brainradioactivity was measured as decays per min (DPM)/μl of serum (afterthe ascorbic acid or sucrose was solubilized from the cells in thepresence of 70% methanol) which was equivalent to the integral of theplasma radioactivity. The BBB PS product is calculated:

% injected dose/gm of brain tissue=PS×AUC

[0090] where the variables are defined, as follows: t=time$V_{D} = \frac{\frac{\left\lbrack {}^{14}{C - {{AA}\quad {or}\quad {DHA}}} \right\rbrack {dpm}}{{gm}\quad {brain}\quad {tissue}}({brain})}{\frac{\left\lbrack {}^{14}{C - {{AA}\quad {or}\quad {DHA}}} \right\rbrack {dpm}}{{\mu l}\quad {serum}}\left( {{external}\quad {organ}} \right)}$$V_{0} = \frac{\frac{\left\lbrack {}^{3}{H - {Sucrose}} \right\rbrack {dpm}}{{gm}\quad {brain}\quad {tissue}}({brain})}{\frac{\left\lbrack {}^{3}{H - {Sucrose}} \right\rbrack {dpm}}{{\mu l}\quad {serum}}\left( {{external}\quad {organ}} \right)}$

[0091] The rats were anesthetized with a mixture of ketamine 90 mg/kgand xylazine 10 mg/kg anesthesia during the procedure. The xylazinecauses a hyperglycemia and hypoinsulinemia in the animals with the serumglucose measured at approximately 280 mg/dl 30 min after induction ofanesthesia (24,25). This is almost three-fold higher than baselineglucose concentrations in the rats and affects transport through GLUT1and therefore the PS calculations. Radiolabeled test compound(³H-sucrose, ¹⁴C-ascorbic acid, ¹⁴C-dehydroascorbic acid) was injectedinto a cannulated femoral vein in groups of 3 rats. Sucrose was used asa V₀ marker (plasma volume marker). For 30 seconds (t) after injectionarterial blood was collected by gravity from a catheter cannulated inthe abdominal aorta and then the animal was sacrificed and the brainharvested.

[0092] Results and Discussions

[0093] Mice and rats were injected into the tail vein with ¹⁴C-ascorbicacid, ¹⁴C-dehydroascorbic acid or ³H-sucrose. Three min afterintravenous injection the animals were sacrificed, the brains harvestedand the methanol soluble fraction counted by liquid scintillation.Approximately 4% of the dehydroascorbic acid (expressed as percent ofinjected dose (ID) per gram of brain tissue) was found in the brainafter 3 min (FIGS. 1A and 1B). Injected ascorbic acid and sucroseyielded only trace radioactivity in the brain homogenate at 3 min,indicating that ascorbic acid could not pass the BBB. Because sucrose isnot metabolized or transported it is used as a marker of plasma volume(12). The small amount of radioactivity present in the brain of thesucrose and ascorbic acid-injected animals was consistent with theradioactivity being present within the brain blood vessels.High-performance liquid chromatography (HPLC) analysis of the methanol(70%) fraction of the brain homogenate showed that the form of thevitamin C accumulated in the brain of dehydroascorbic acid-injectedanimal was >85% ascorbic acid (FIG. 1C). This result indicated thatdehydroascorbic acid was transported across the BBB and retained asascorbic acid in the brain.

[0094] Brain radioactivity, after dehydroascorbic acid injection,reached a maximum of 4.3% of ID/gram brain tissue at 3 min, decreased to3.3% at 25 min, and remained at that level for up to 2 hours afterinjection (FIG. 1D, 1E). Injection of sucrose and ascorbic acid resultedin a maximum brain accumulation of 0.4% ID/gram brain tissue at 15 to 30seconds after injection (FIG. 1D). Brain radioactivity in thesucrose-injected animals decreased to <0.1% after 15 min, concomitantwith the fall in serum radioactivity in these mice (FIG. 1E, 1G). Inascorbic acid-injected mice there was an increase in brain radioactivityto 1.1% ID/gram brain tissue 2 hours after injection, a time periodduring which there was a decreasing amount of radioactivity in the serum(FIGS. 1E, 1G). The serum radioactivity concentration at 15 secondsafter dehydroascorbic acid injection was 8% ID/gram serum, whereas thecorresponding figure in mice injected with ascorbic acid was 27%. Thusdehydroascorbic acid was cleared from the circulation substantiallyfaster than ascorbic acid (FIG. 1F). At the 3-min time point theradioactivity in the serum of the ascorbic acid and dehydroascorbicacid-injected animals was equivalent (FIG. 1G). Radioactivity remainingin the serum of the dehydroascorbic acid-injected animals at 5 min wasassociated with ascorbic acid (FIG. 1H).

[0095] Injected ¹⁴C-ascorbic acid showed no measurable transport intothe brain over the first 30-min, but some radioactivity accumulated inthe brain at longer time periods. There are at least three potentialexplanations for this result. The first is that the ascorbic acid wasmetabolized in the interval time period and the counts in the brainrepresented transported radiolabeled metabolic breakdown products ofascorbic acid. Such an explanation is unlikely as the HPLC resultsdemonstrated that the majority of the radioactivity in thedehydroascorbic acid-injected brain was eluted in radioactive peaksconsistent with intact ascorbic acid. A second possibility is thepresence of a small number of Na⁺-ascorbate cotransporters at the BBB orchoroid plexus, which is unlikely since the accumulation of ascorbicacid did not occur linearly with time, as it would in this case, butonly occurred after 30 min (13). The interpretation is that oxidation ofascorbic acid in the microenvironment occurred in vivo leading to theproduction of dehydroascorbic acid which was then transported across theBBB and retained in the brain as ascorbic acid.

[0096] The serum concentration of injected dehydroascorbic acid reachedonly 20 to 25% of the serum concentration of ascorbic acid or sucroseduring the initial several minutes after injection. Sucrose has notransport mechanism, therefore its clearance from the serum was slow.Part of the clearance mechanisms for ascorbic acid and dehydroascorbicacid are through transport, the GLUTs in the case of dehydroascorbicacid and potentially a Na⁺-ascorbate cotransporter in the case ofascorbic acid (4). The rapid clearance of dehydroascorbic acid from theserum likely reflected the large number of glucose transportersavailable for transport.

[0097] The glucose transporter GLUT1 selectively transports D-glucosebut not L-glucose. In order to confirm that dehydroascorbic acid passedthe BBB through GLUTS, inhibition experiments were conducted with D- andL-glucose. 2-Deoxy-D-glucose (D-deoxyglucose) and D-glucose (data notshown) inhibited uptake of dehydroascorbic acid in the brain in adose-dependent fashion up to 70%, whereas L-glucose and leucine had noeffect (FIG. 2A). The uptake of leucine, which is not transported byGLUTs, but crosses the BBB largely through L system transporters and toa minor extent by the ASC system transporter (14), was not affected byincreasing concentrations of L-glucose of D-deoxyglucose (FIG. 2B) norwere the serum concentrations of ascorbic acid, dehydroascorbic acid andleucine affected by increasing concentrations of D-deoxyglucose orL-glucose (data not shown). These results established thatD-deoxyglucose inhibits dehydroascorbic acid from entering the brainthrough the glucose transporters but does not affect certain othertransport systems or alter general BBB permeability by osmotic effects.

[0098] The external organ approach, utilizing serum as the externalorgan, was used to calculate the BBB permeability-surface areas product(PS) in the Fischer F344 rat (15). The calculated PS of¹⁴C-dehydroascorbic acid was 136±12 (SEM) μl/min/gm brain tissue,¹⁴C-ascorbic acid was −0.44±0.24 μl/min/gm brain tissue, and³H-D-deoxyglucose was 44±3.2 μl/min/gm brain tissue. The difference inthe BBB permeability-surface area products (PS) between ascorbic acidand dehydroascorbic acid illustrated the marked differences in the BBBtransport between the redox states of vitamin C. The calculated PS ofascorbic acid was approximately 0 μl/min/gm brain tissue at 30 seconds,similar to sucrose, which indicates no transport across the BBB. The PSof dehydroascorbic acid was 3-fold greater than D-deoxyglucose whichcorresponds with the difference in the K_(m) values between the twocompounds. The apparent K_(n) of D-deoxyglucose for transport was 2.5 mMin HL60 cells compared with an apparent K_(m) of 0.85 mM fordehydroascorbic acid in HL60 cells (2,3).

[0099] Digital autoradiography of the brain of a rat injected with¹⁴C-dehydroascorbic acid and a rat injected with 14C-ascorbic acid wasperformed to confirm the anatomical distribution of the injectedcompounds (FIG. 3).

[0100] Autoradiographic evidence of activity accumulation in the brainwas seen only in animals injected with dehydroascorbic acid. ¹⁴C-sucrosewas used as a marker of intravascular volume.

[0101] The results of this study established that the transport ofvitamin C into the brain is mediated by GLUTs at the BBB which transportdehydroascorbic acid. Ascorbic acid itself is not transportable acrossthe BBB. The glucose transport in vivo therefore was found to functioncomparably to in vitro models in that only the oxidized form of vitaminC, dehydroascorbic acid, was transportable (1-3). Dehydroascorbic acidwas reduced to ascorbic acid after passing the BBB and was retained inthe brain as ascorbic acid. This trapping mechanism allows for theaccumulation of higher concentrations of vitamin C in the brain than inthe blood. Overall, the findings point to the oxidation of ascorbic acidas being the critical step in the regulation of the accumulation ofvitamin C in the brain.

[0102] The current recommended daily allowance of vitamin C is 60 mgdaily and yields a steady-state plasma concentration of approximately 24μM in human volunteers (16). Only ascorbic acid is detected in theserum, with dehydroascorbic acid at trace serum levels or not measurable(17). The vitamin C injected in this study was approximately 500 μM,which is 5-fold greater than the physiologic serum concentration ofvitamin C in rodents (18). In this study, at physiologic glucoseconcentrations, dehydroascorbic acid transport through GLUT1 did occur.The serum concentration of glucose in normal rodents is approximately 10mM yet there is still dehydroascorbic acid transport to the brainindicating that both dehydroascorbic acid and glucose are substrates ofthe GLUTs under physiologic conditions. This result is consistent within vitro data demonstrating that a deoxyglucose concentration greaterthan 50 mM is necessary to block the transport of dehydroascorbic acidthrough GLUT1 (2,3).

[0103] James Lind detailed the clinical description of scurvy in ATreatise of the Scurvy in 1772. He concluded his report of the autopsyresults of scorbutic patients' “ravaged bodies” as follows, “What wasvery surprising, the brains of those poor creatures were always soundand entire . . . ” (19). There thus appeared to be a mechanism for theaccumulation and storage of ascorbic acid in the brain such that thebrain would be the last organ depleted of vitamin C. The normal humanbrain has a vitamin C concentration of approximately 1 mM, 10 times thenormal serum concentration (20). The precise role of vitamin C in thebrain is uncertain, but ascorbic acid may be a cofactor of dopamineβ-hydroxylase and is thus involved in the biosynthesis ofcatecholamines. Vitamin C can also inhibit the peroxidation of membranephospholipids and act as a scavenger of free radicals in the brain(21,22). The results of this study demonstrate the physiologicalimportance of vitamin C transport through GLUT1 in the form ofdehydroascorbic acid and define the mechanism by which the brain obtainsand retains vitamin C.

[0104] Recent data show that large quantities of vitamin C can be loadedinto the brain. An experiment was done in which the carotid artery of asubject rat was cannulated with a catheter and 24 mg of dehydroascorbicacid was injected into the artery. The injected dehydroascorbic acid wasspiked with a tracer amount of radioactive (¹⁴C-labeled) dehydroascorbicacid. The dehydroascorbic acid was infused over forty minutes and thebrain was harvested. The amount of radioactive vitamin C was quantitatedin the brain and total amount of injected vitamin C that accumulated inthe brain was thus extrapolated. The experiment demonstrated that 2.6 mgof vitamin C accumulated in the brain of the subject rat during theforty minute injection period, which was approximately 11% of theinjected dose. This shows that it is possible to achieve pharmacologicconcentrations of vitamin C in the brains of subject animals. It is ofnote that the total vitamin C concentration in the normal adult ratbrain is approximately 150 μg. A log-fold greater Vitamin C thanbaseline normal concentration of Vitamin C was thus achieved.

REFERENCES AND NOTES

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[0123] 19. Stewart, C. P. & Guthrie, D. Lind's Treatise on Scurvy(Edinburgh University Press, Edinburgh, ed. 1, 1953).

[0124] 20. Hornig, D. Ann. N.Y. Acad. Sci. 258, 103-118 (1975).

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[0142] Second Series of Experiments

[0143] Although stroke is the leading cause of permanent morbidtyworldwide (1), current therapy is limited to thrombolysis, with a narrowtherapuetic window and requirement for sophisticated pretreatmentimaging. (2,3) Acute restoration of flow can also lead to the productionof reactive oxygen species, which are directly toxic to neurons andglia, and may exacerbate leukocyte accumulation, (4) microvascularthrombosis, and nitric-oxide mediated injury. (5-7) Efforts to abrogateoxidant stress are complicated by the limited ability of antioxidants tocross the blood brain-barrier. (9) Previous work has defined thedehydroascorbic acid (DHA)-GLUT1 transport mechanism by which cellsaccumulate and retain vitamin C, (9, 17) a potent anitoxidant known toquench free radicals in the setting of ischemia. (10) More recently, therapid transport of DHA across the blood-brain barrier and its retentionin the brain as ascorbic acid was described in rodents. (11) Here, wedescribe the ability of DHA, given intravenously in the setting ofmurine stroke to improve cerebral blood flow, functional outcome, andthe volume of infarcted brain tissue. The level of protection achievedis far greater than that seen with ascorbic acid administration,supporting our hypothesis that the use of a potent antioxidant, withclearly defined blood brain-barrier penetrability, may have a role inthe treatment of thromboembolic stroke in humans.

[0144] Radiolabeled dehydroascorbic acid (DHA) was generated byincubating ¹⁴C-ascorbic acid with ascorbate oxidase, 1 unit/1.0 mmolL-ascorbate (derived from Cucurbita species, Sigma). Dithiothreitol (0.1mmol/L) was then added separately to both DHA and ascorbate prior to useas a reducing agent. For experiments examining the effect of focalcerebal ischemia on the DHA's ability to: (1) cross the blood-brainbarrier and, (2) protect cerebral tissue, we employed an intraluminalmurine model of reversible (45 minutes) or permanent (24 h) right middlecerebral artery occlusion. (12) Anesthetized normothermic C57/6J BL micewere treated prior to ischemia via penile injection with either highdose DHA (250 mg/kg), low dose DHA (40 mg/kg), ascorbate (250 mg/kg), orsucrose (a non-metabolized, non-transportable marker of plasma volume).All animals underwent transcranial laser doppler measurement of corticalcerebral blood flow perioperatively. Nine mice were subjected to 10minutes or two hours of focal ischemia and immediately sacrificed inorder to assess DHA (n=3), ascorbate (n=3), and sucrose (n=3) transportas measured by radiation scintillation counting using 5 μCi of¹⁴C-ascorbic and (L-[1-¹⁴C]-ascorbic acid, specific activity, 6.6mCi/mmol, Dupont NEN), ¹⁴C-DHA or ³H-sucrose ([fructose-1-³H]-sucrose,specific activity 20.0 Ci/mmol., Dupont NEN) as previously described.(11) In contrast, the experimental cohorts were followed for 24 hoursand underwent pre-sacrifice neurological examination, as previouslydescribed. 13 Infarct volumes were calculated by digital analysis ofserial, 2,3,5-triphenyltetrazolium-stained, sections, with volumesexpressed as percentage of the ipsilateral hemisphere.¹² There were 67animals in the reperfusion cohort (including vehicle, 40 mg/kg DHA, 250mg/kg DHA, and 250 mg/kg ascorbate cohorts) and 54 animals in thenonreperfused cohort (including vehicle, 250 mg/kg DHA, and 250 mg/kgascorbate cohorts) Comparisons were made between groups with atwo-tailed Student t-test for unpaired variable. All studies wereperformed in accordance with an institutionally approved animalprotocol.

[0145] Tracer studies revealed accumulation of nearly 4% of the DHA(expressed as percent of injected dose (TD) per gram of brain tissue)compared to only trace levels of ascorbic acid and sucrose in ischemicanimals, a finding not significantly different from that seen insham-operated animals (FIG. 1). Previous studies demonstrated that theform of vitamin C accumulating in the brains of DHA-injected animalsis >85% ascorbic acid as a result of reduction of transported DHA. (11)Both high (250 mg/kg) and low dose (40 mg/kg) DHA pre-treatment ofanimals undergoing reperfused stroke, demonstrated a dose-dependentimprovement in post-ischemic cerebral perfusion compared to both sucroseand ascorbic acid treated animals (19±3% vehicle, 13±1% AA, 28±2%low-DHA, 40±4% high-DHA; p<0.05 for either DHA vs. either control) (FIG.2A). In addition, DHA conferred dose-dependent cerebroprotection asevidenced by both decreased cerebral infarct volumes (54±6% vehicle,58±4% AA, 22±4% low-DHA, 12±4% high-DHA; p<0.05 for either DHA vs.either control) (FIG. 2B) and reductions in neurological deficit scores(4.0±0.2 vehicle, 2.7±0.7 AA, 1.6±0.2 low-DHA, 1.9±0.2 high-DHA; p<0.05for either DHA vs. vehicle) (FIG. 2C). In contrast, ascorbic acidtreatment was not associated with significant improvements in eitherinfarct size or neurological function, nor did ascorbic acid treatmentreduce overall mortality as was the case with DHA (FIG. 2D). Ascorbicacid treated animals died at nearly twice the rate of the DHA-treatedcohort (60% vehicle, 50% AA, 24% low-DHA, 27% high-DHA; p<0.05 forlow-DHA vs. vehicle).

[0146] Since clinical therapeutic antioxidant strategies might beemployed prior to hospitalization and reperfusion, we examined theeffect of high-dose DHA on non-reperfused stroke as well. As in theprior set of experiments, high-dose DHA improved pre-sacrifice regionalcerebral cortical perfusion whereas ascorbic acid did not (13±2% vehicle15±3% AA, and 30±3% high-DHA; p<0.05 for DHA vs. either control). Thisimproved perfusion was associated with similar reductions in infarctvolume (51±6% vehicle, 46±6% AA, and 26±5% high-DHA, p<0.01 vs.vehicle), and sacrifice neurological deficit scores (3.0±0.3% vehicle,3.2±0.4% AA, and 1.9±0.2% high-DHA; p<0.005 for DHA vs. either control).As in the setting of reperfusion, DHA reduced mortality by nearly 50%(50% vehicle, 64% AA, and 30% high-DHA.

[0147] Therapeutic, non-enzymatic scavenging of free radicals can beaccomplished by ascorbic acid but only at supraphysiologicconcentrations. (10) We confirmed previous findings that intravenousadministration of DHA allows supraphysiologic concentrations ofascorbate to be achieved in the brain whereas ascorbic acidadministration does not. (11) Importantly, our results demonstrate thein vivo cerebroprotection conferred by DHA in the setting of bothtransient and permanent, focal cerebral ischemia. In doing so these datafurther implicate the role of ascorbate in free radical scavengingfollowing cerebral ischemia, (14, 15) and underscore the importance ofpharmacologically increasing cerebral vitamin C concentrations followingcerebral ischemia. (14,16) These data also support the notion that localascorbate may be critical in limiting nitric-oxide signaling failuremediated by superoxide, since DHA had a dose-dependent effect oncerebral blood flow. (10) Maintenance of microvascular potency bystabilizing NO-dependent vascular signalling requires very high locallevels of ascorbate, since the reaction of ascorbate with superoxide is10⁵-fold slower than that of superoxide with SOD and certainly NO.Together with previous work this study also suggests that the 10-100mmol/L concentrations of acrorbate predicted to inhibitsuperoxide-dependent vasoconstriction can be achieved with theadministration of DHA but not ascorbic acid. (10,11) DHA's ability toimprove perfusion in ischemic beds subjected to both transient andpermanent ischemia has major implications for the treatment ofprogressive microvascular failure in the setting of clinical stroke.

[0148] References for Second Series of Experiments

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What is claimed:
 1. A method for increasing the concentration ofascorbic acid in a cell which comprises contacting the cell with anamount of dehydroascorbic acid effective to increase the concentrationof ascorbic acid in the cell.
 2. A method for increasing the antioxidantpotential of a cell which comprises contacting the cell with an amountof dehydroascorbic acid effective to increase the antioxidant potentialof the cell
 3. The method of claim 1 or 2, wherein the cell is a braincell.
 4. The method of claim 1 or 2, wherein the cell is present in atissue.
 5. The method of claim 4, wherein the tissue is a brain tissue.6. The method of claim 1 or 2, wherein the cell is present in a subjectand the contacting is effected by administering the dehydroascorbic acidto the subject.
 7. The method of claim 6, wherein the subject is human.8. A method for increasing the concentration of ascorbic acid in thecells of a subject which comprises administering to the subject anamount of dehydroascorbic acid effective to increase the concentrationof ascorbic acid in the subject's cells.
 9. A method for increasing theantioxidant potential of the cells of a subject which comprisesadministering to the subject an amount of dehydroascorbic acid effectiveto increase the antioxidant potential of the subject's cells.
 10. Amethod for increasing the concentration of ascorbic acid in brain tissueof a subject which comprises administering to the subject an amount ofdehydroascorbic acid effective to increase the concentration of ascorbicacid in the subject's brain tissue.
 11. A method for increasing theantioxidant potential of brain tissue of a subject which comprisesadministering to the subject an amount of dehydroascorbic acid effectiveto increase the antioxidant potential of the subject's brain tissue. 12.The method of claim 3 or 4, wherein the cells are brain cells.
 13. Themethod of claim 3 or 4 or 5 or 6, wherein the subject is a human. 14.The method of claim 6, wherein the subject has a neurodegenerativedisease.
 15. The method of claim 14, wherein the neurodegenerativedisease is Alzheimer's Disease or Parkinson's Disease.
 16. The method ofclaim 6, wherein the subject has neurovascular disease.
 17. The methodof claim 16, wherein the neurovascular disease is stroke.
 18. The methodof claim 6, wherein the subject is afflicted with a genetic disease ofthe nervous system.
 19. The method of claim 18, wherein the geneticdisease of the nervous system is Huntington's Disease.
 20. The method ofclaim 6, wherein the subject is afflicted with a disease which involvesthe oxidative modification of low-density lipoprotein or lipidperoxidation.
 21. The method of claim 20, wherein the disease is stroke,atherosclerosis or a neurodegenerative disorder.
 22. The method of claim6, wherein the subject is afflicted with a behavioral disorder.
 23. Themethod of claim 22, wherein the behavioral disorder is dysthymia,involution depression, aggressiveness via dominance, hyperactivity,deprivation syndrome, separation anxiety, intermittent anxiety,instrumental sociopathy, stereotypies, phobia or a socializationdisorder.
 24. A method for treating or preventing dementia in a subjectcomprising administering to the subject an amount of dehydroascorbicacid effective to increase the concentration of ascorbic acid in thesubject's brain tissue so as to thereby treat or prevent dementia in thesubject.
 25. A method for treating or preventing dementia in a subjectcomprising administering to the subject an amount of dehydroascorbicacid effective to increase the antioxidant potential of the subject'sbrain tissue so as to thereby treat or prevent dementia in the subject.26. A method for treating or preventing diseases involving free radicalsin a subject comprising administering to the subject an amount ofdehydroascorbic acid effective to increase the concentration ofabscorbic acid in the subject's cells so as to thereby treat or preventdiseases involving free radicals in the subject.
 27. A method fortreating or preventing diseases involving free radicals in a subjectcomprising administering to the subject an amount of dehydroascorbicacid effective to increase the antioxidant potential of the subject'scells so as to thereby treat or prevent diseases involving free radicalsin the subject.
 28. The method of claim 27, wherein the disease iscancer, a cardiovascular disease or cataracts.
 29. A method for slowingthe aging process of a subject comprising administering to the subjectan amount of dehydroascorbic acid effective to increase the antioxidantpotential of cells so as to thereby slow the aging process in a subject.30. A method for treating a subject infected with human immunodeficiencyvirus comprising administering to the subject an amount ofdehydroascorbic acid effective to treat the subject infected with humanimmunodeficiency virus.
 31. The method of claim 6, wherein thedehydroascorbic acid is administered orally, intravenously,subcutaneously or intramuscularly.
 32. A method for treating orpreventing a neurodegenerative disease in a subject comprisingadministering to the subject an amount of dehydroascorbic acid effectiveto increase the concentration of ascorbic acid in the subject's braintissue so as to thereby treat or prevent a neurodegenerative disease inthe subject.
 33. A method for treating or preventing a neurodegenerativedisease in a subject comprising administering to the subject an amountof dehydroascorbic acid effective to increase the antioxidant potentialof the subject's brain tissue so as to thereby treat or prevent aneurodegenerative disease in the subject.
 34. The method of claim 32 or33, wherein the neurodegenerative disease is Alzheimer's Disease orParkinson's Disease.
 35. A method for treating or preventing stroke orneurovascular disease in a subject comprising administering to thesubject an amount of dehydroascorbic acid effective to increase theconcentration of ascorbic acid in the subject's brain tissue so as tothereby treat or prevent neurovascular disease in the subject.
 36. Amethod for treating or preventing stroke or neurovascular disease in asubject comprising administering to the subject an amount ofdehydroascorbic acid effective to increase the antioxidant potential ofthe subject's brain tissue so as to thereby treat or prevent stroke orneurovascular disease in the subject.
 37. A method for treating orpreventing a behavioral disorder in a subject comprising administeringto the subject an amount of dehydroascorbic acid effective to increasethe concentration of ascorbic acid in the subject's brain tissue so asto thereby treat or prevent a behavioral disorder in the subject.
 38. Amethod for treating or preventing a behavioral disorder in a subjectcomprising administering to the subject an amount of dehydroascorbicacid effective to increase the antioxidant potential of the subject'sbrain tissue so as to thereby treat or prevent a behavioral disorder inthe subject.
 39. The method of claim 37 or 38, wherein the behavioraldisorder is dysthymia, involution depression, aggressiveness viadominance, hyperactivity, deprivation syndrome, separation anxiety,intermittent anxiety, instrumental sociopathy, stereotypies, phobia or asocialization disorder.
 40. The method of claim 6 further comprisingadministering to the subject a therapeutically effective amount of asecond agent.